We propose a series of studies of T lymphocyte activation in old and young mice aimed at identifying in biochemical terms the defect(s) that prevent many T cells from old mice from responding to mitogens. We have previously shown age-related derangements in the generation of cytoplasmic calcium signals in T cells from old donors, and now propose to test two explanatory hypotheses: (a) that aged T cells have over-active calcium extrusion systems; and (b) that the low resting membrane potential of T cells from old mice interferes with calcium signal generation. Our studies of early signal transduction have hinted that pathways dependent on protein kinase C may be less impaired by aging than other kinase-dependent paths; we now propose more direct and systematic tests of protein kinase function and substrate specificity in T cells from old mice. We have previously documented age-related defects in Con A-induced expression of c-myc mRNA. We now propose to test for age-related changes in expression of selected cell cycle-related genes (c-fos, c-jun, IL-2, and IL-2R) in responses to mitogenic stimuli (Con A, anti-CD3). Nonmitogenic activators (PMA, ionomycin) will be used in studies of gene expression and protein kinase function to identify pathways particularly susceptible to age-related dysfunction. Antibodies to c-myc and c-fos proteins will be used to look for altered gene expression at the single cell level and in T cell subsets thought to be particularly susceptible to senescent change. Finally we propose to look for evidence of age-related defects in splicing of primary RNA transcripts, defects that we have previously suggested may be responsible for age-related loss of myc mRNA accumulation. We hope to identify specific age-related defects in calcium signals, protein kinase function, and gene expression that might contribute to poor T lymphocyte function in old mice. Grant=R01AG071140004 As animals age, their adipocytes become less responsive to lipolytic and lipogenic signals. The underlying mechanism for this loss of responsiveness is unknown. Current data suggest that this decline cannot be accounted for solely by alterations in specific cell surface receptors. We hypothesize that the age-related decline in adipocyte responsiveness stems from aberrant regulation of specific gene expression. We will use both mice and the mouse 3T3F442A cell line to investigate and compare in vivo and in vitro aging. 3T3F442A cells differentiate into adipocytes in a manner that is strikingly similar to what occurs during normal mouse development and have the advantage of living for several months in culture. We will examine genes which control adipose gene expression (c-fos, c-jun, C/EBP) as well as several adipocyte-specific genes (glycerophosphate, dehydrogenase, adipsin, aP2). We will determine if there are global or specific age-related alterations in fat cell gene expression by comparing the mRNA levels of these 6 genes in adipose tissue from mice at different ages. We will also determine whether any alterations in gene expression are prevented or reversed by diet. We will measure the levels of these same mRNAs in 3T3-adipocytes over a 3 month period to compare the effects of aging in vivo and in vitro. The response of young and old 3T3- adipocytes to lipolytic and lipogenic agents will be tested to see if there is an age-related decline in responsiveness in vitro. Finally, we will determine whether age-related changes in gene expression are due to changes in the levels of transcription, mRNA stability, and/or posttranscriptional processing. The results will provide important insights into the molecular events that accompany the aging process in a well defined differentiated cell type.